446 APPENDIX 



:h square 





four hours, then placing a small piece of the bone (not exceeding ^ inch sqi 

 and % inch thick) in concentrated sulphurous acid. 



This decalcifies in about two to seven days. Wash thoroughly in alkaline water 

 and then in tap water. Pass through alcohols and xylol and imbed and section as 

 before described. 



To Stain Sections. It is first necessary to affix the section to a slide or cover-glass 



To attach the section firmly to the slide, so that it will not become detached in 

 subsequent treatment, pick up a section on a strip of cigarette paper. 



A sheet of cigarette paper is cut into about five pieces (% X iM inch). In- 

 serting the strip of cigarette paper under the section, it is easily lifted up out of 

 the water. Then apply the slip of cigarette paper, section downward, to a perfectly 

 clean slide. Blot with a piece of filter-paper, then strip off the piece of filter-paper 

 leaving the section smoothly applied to the slide. Next place in the 37C. incubator 

 for twelve to twenty-four hours and the section will be found to be so firmly attached 

 that it will not be dislodged by subsequent treatment. 



For Immediate Diagnosis. Take a very small loopful of albumin fixative (white 

 of fresh egg, 50 c.c., glycerine, 50 c.c.; sodium salicylate, i gram) and deposit it on a 

 cover-glass. Now take up a loopful of 30% alcohol (i drop of 95% alcohol and 2 

 drops of water) and applying it over the albumin fixative, smear out the mixture 

 uniformly over the cover-glass. 



2. Pick up a section on a strip of cigarette paper and apply it to the prepared sur- 

 face on the cover-glass. Blot with gentle pressure with a piece of filter-paper over 

 the strip of cigarette paper, and strip off this latter, leaving the section attached to 

 the cover-glass. 



3. Now, turning the flame of the Bunsen burner down very low or with a small 

 alcohol flame, we hold the cover-glass in a Stewart's forceps, section side up, over 

 the flame and slowly lower it until the paraffin is observed to melt. This shows a 

 temperature of about 5oC. The section is fixed by the coagulation of the albumin 

 at about 7oC. To obtain this temperature lower the cover-glass still more, and the 

 moment vapor is seen to rise from the section it indicates the attachment of the sec- 

 tion to the cover-glass. 



4. Flood section on cover-glass or slide with xylol; this dissolves out the paraffin. 

 It is better to pour off the first xylol and drop on fresh xylol (one minute). 



5. Remove xylol with two applications of absolute alcohol (one minute). 



6. Treat specimen with two or three applications of 95% alcohol (one to two 

 minutes). 



7. Next wash in water (one to two minutes). 



8. Flood specimen with haemalum or Delafield's haematoxylin (three to seven 

 minutes). 



9. Wash in tap water for about two to five minutes until a purplish tinge is devel- 

 oped hi the section. The alkali in ordinary tap water develops this color. 



10. Apply i to 1000 eosin (aqueous) for thirty seconds to one minute. 



11. Wash in water; then in 95% alcohol; then in absolute alcohol. 



12. Apply a few drops of xylol and as soon as the section is perfectly transpa 

 mount in balsam, or immersion oil. 



The staining by haematoxylin and eosin is the best for the study of the histology 

 of a section. It only requires about ten minutes to run a preparation through for 

 diagnosis by this method. 



